Bottom Line:
We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay.The final data set contained 166 interactions between 10 viral proteins and 122 human proteins.Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively.

Background: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.

Results: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.

Conclusions: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

Figure 3: Effect of SPG21 and FANCG knockdown on viral promoter activation. Jurkat-LTR-Luc cells were transduced with lentiviral particles expressing a control shRNA and three validated shRNAs targeting various sequences of the SPG21 and FANCG mRNAs. Cells were cultured for 24 hours, and luciferase activities were determined from cell lysates and normalized to corresponding cell viability data (measured by WST1 test). Results are means of three experiments and error bars indicate standard errors.

Mentions:
Many HTLV-human interactions in our data set (106/166) involved the retroviral transactivator proteins HTLV-1 Tax (57/166) or HTLV-2 Tax2 (49/166). To examine the functional consequences of these associations, HEK293T cells were cotransfected with expression vectors for Tax-1 and Tax-interacting proteins, together with a firefly luciferase reporter driven by the HTLV-1 LTR promoter. As determined by normalized luciferase reporter assays, we identified 31 proteins (37% of the 83 Tax-interacting proteins) that regulated HTLV-1 LTR promoter activation by Tax (Figure 2 and Additional file 1: Table S3). There were 8 host factors that significantly enhanced Tax transactivation activities suggesting their potential implication in viral replication and persistence in infected cells. Another group of 23 cellular proteins down-regulated HTLV-1 LTR viral promoter activation and as such may be implicated in the viral latency which allows viruses to escape immune surveillance (Figure 2 and Additional file 1: Table S3). We selected two Tax1-cellular partners, SPG21, involved in the repression of T cell activation [22], and FANCG, a DNA damage response activated protein [23-25], for further validation in a T lymphocyte cell line. We used Jurkat T cells harboring a HTLV-1 LTR luciferase reporter (Jurkat-LTR-Luc) to confirm potential roles of SPG21 and FANCG in viral replication. We transduced Jurkat-LTR-Luc cells with a control shRNA and three validated shRNAs directed against SPG21 or FANCG and measured luciferase reporter-expression and cell viability. In accordance with regulation of Tax-transactivation data (Figure 2 and Additional file 1: Table S3), knockdown of SPG21 increased HTLV-1 LTR promoter activity while depletion of FANCG decreased HTLV-1 LTR promoter activity (Figure 3).

Figure 3: Effect of SPG21 and FANCG knockdown on viral promoter activation. Jurkat-LTR-Luc cells were transduced with lentiviral particles expressing a control shRNA and three validated shRNAs targeting various sequences of the SPG21 and FANCG mRNAs. Cells were cultured for 24 hours, and luciferase activities were determined from cell lysates and normalized to corresponding cell viability data (measured by WST1 test). Results are means of three experiments and error bars indicate standard errors.

Mentions:
Many HTLV-human interactions in our data set (106/166) involved the retroviral transactivator proteins HTLV-1 Tax (57/166) or HTLV-2 Tax2 (49/166). To examine the functional consequences of these associations, HEK293T cells were cotransfected with expression vectors for Tax-1 and Tax-interacting proteins, together with a firefly luciferase reporter driven by the HTLV-1 LTR promoter. As determined by normalized luciferase reporter assays, we identified 31 proteins (37% of the 83 Tax-interacting proteins) that regulated HTLV-1 LTR promoter activation by Tax (Figure 2 and Additional file 1: Table S3). There were 8 host factors that significantly enhanced Tax transactivation activities suggesting their potential implication in viral replication and persistence in infected cells. Another group of 23 cellular proteins down-regulated HTLV-1 LTR viral promoter activation and as such may be implicated in the viral latency which allows viruses to escape immune surveillance (Figure 2 and Additional file 1: Table S3). We selected two Tax1-cellular partners, SPG21, involved in the repression of T cell activation [22], and FANCG, a DNA damage response activated protein [23-25], for further validation in a T lymphocyte cell line. We used Jurkat T cells harboring a HTLV-1 LTR luciferase reporter (Jurkat-LTR-Luc) to confirm potential roles of SPG21 and FANCG in viral replication. We transduced Jurkat-LTR-Luc cells with a control shRNA and three validated shRNAs directed against SPG21 or FANCG and measured luciferase reporter-expression and cell viability. In accordance with regulation of Tax-transactivation data (Figure 2 and Additional file 1: Table S3), knockdown of SPG21 increased HTLV-1 LTR promoter activity while depletion of FANCG decreased HTLV-1 LTR promoter activity (Figure 3).

Bottom Line:
We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay.The final data set contained 166 interactions between 10 viral proteins and 122 human proteins.Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively.

Background: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.

Results: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.

Conclusions: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.